Methods for the Production of Products in Host Cells

ABSTRACT

The invention provides methods and host cells for the production of ascorbic acid intermediates. The invention also provides host cells having a modification in a polynucleotide that uncouples the catabolic pathway from the oxidative pathway by deleting the encoding for an endogenous enzymatic activity that phosphorylates D-glucose at its 6th carbon and/or a polynucleotide that has deleted the encoding for endogenous enzymatic activity that phosphorylates D-gluconate at its 6th carbon. Such host cells are used for the production of products, such as, ascorbic acid intermediates. Nucleic acid and amino acid sequences with inactivated enzymatic activity which phosphorylates D-glucose at its 6th carbon and inactivated enzymatic activity which phosphorylates D-gluconate at its 6th carbon are provided.

This application is a Continuation of U.S. patent application Ser. No.11/282,000 filed Nov. 17, 2005 which is a Continuation of applicationSer. No. 10/116,821, filed Apr. 4, 2002 which claims the benefit ofprovisional application Ser. No. 60/282,277, filed Apr. 5, 2001 andclaims the benefit of provisional application Ser. No. 60/281,571, filedApr. 4, 2001.

STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH

This invention was made with the United States Government support underAward No. 70 NANB 5H1138 awarded by the United States Department ofCommerce. The Government has certain rights in this invention.

TECHNICAL FIELD

The present invention relates to engineering of metabolic pathways inhost cells and provides methods and systems for the production ofproducts in host cells. In particular, the present invention providesmethods and systems for the production of ascorbic acid intermediates inhost cells.

BACKGROUND ART

Numerous products of commercial interest, such as intermediates ofL-ascorbic acid, have been produced biocatalytically in geneticallyengineered host cells. L-Ascorbic acid (vitamin C, ASA) finds use in thepharmaceutical and food industry as a vitamin and antioxidant. Thesynthesis of ASA has received considerable attention over many years dueto its relatively large market volume and high value as a specialtychemical.

The Reichstein-Grussner method, a chemical synthesis route from glucoseto ASA, was first disclosed in 1934 (Helv. Chim. Acta 17:311-328).Lazarus et al. (1989, “Vitamin C: Bioconversion via a Recombinant DNAApproach”, Genetics and Molecular Biology of Industrial Microorganisms,American Society for Microbiology, Washington D.C. Edited by C. L.Hershberger) disclose a bioconversion method for production of anintermediate of ASA, 2-keto-L-gulonic acid (2-KLG, KLG) which can bechemically converted to ASA. This bioconversion of carbon source to KLGinvolves a variety of intermediates, the enzymatic process beingassociated with co-factor dependent 2,5-DKG reductase activity (2,5-DKGRor DKGR).

Many bacterial species have been found to contain DKGR, particularlymembers of the Coryneform group, including the genera Corynebacterium,Brevibacterium, and Arthrobacter. DKGR obtained from Corynebacterium sp.strain SHS752001 is described in Grindley et al. (1988) Applied andEnvironmental Microbiology 54: 1770-1775. DKGR from Erwinia herbicola isdisclosed in U.S. Pat. No. 5,008,193 to Anderson et al. Other reductasesare disclosed in U.S. Pat. Nos. 5,795,761; 5,376,544; 5,583,025;4,757,012; 4,758,514; 5,004,690; and 5,032,514.

Host cells having mutations in enzymes involved in glycolysis have beendescribed. Yeast having mutations in glucokinase are described inHarrod, et al. (1997) J. Ind. Microbiol. Biotechnol. 18:379-383;Wedlock, et al. (1989) J. Gen. Microbiol. 135: 2013-2018; and Walsh etal. (1983) J. Bacteriol. 154:1002-1004. Bacteria deficient inglucokinase have been described. Pediococcus sp. deficient inglucokinase is described in Japanese patent publication JP 4267860.Bacillus sphaericus lacking glucokinase is described in Russell et al.(1989) Appl. Environ. Microbiol. 55: 294-297. Penicillium chrysogenumdeficient in glucokinase is described in Barredo et al.(1988)Antimicrob. Agents-Chemother 32: 1061-1067. A glucokinase-deficientmutant of Zymomonas mobilis is described in DiMarco et al. (1985) Appl.Environ. Microbiol. 49:151-157.

Bacteria which ferment glucose through the Embden-Meyerhof pathway, suchas members of Enterobacteriacea and Vibrionaceae, are described inBouvet, et al. (1989) International Journal of Systematic Bacteriology,p. 61-67. Pathways for metabolism of ketoaldonic acids in Erwinia sp.are described in Truesdell, et al, (1991) Journal of Bacteriology, pp6651-6656.

However, most of the current methodologies utilized to produce compoundsover-express the products. There are still problems remaining concerningthe diversion of substrates used to produce the desired end-product bythe cell for metabolic (catabolic) purposes, reducing the efficiency andoverall production of thereof. Thus, there remains a need for improvedmethods for the production of products through pathways which arecoupled to the metabolic pathways of host cells. The present inventionaddresses that need.

All publications cited herein are hereby incorporated by reference intheir entirety.

SUMMARY OF THE INVENTION

The present invention provides methods to produce ascorbic acidintermediates comprising host cells genetically engineered to reducecarbon substrate diverted to metabolic pathways, thus increasing theproductivity of the host cell.

In one embodiment, the present invention provides a method of enhancingthe production of an ascorbic acid intermediate from a carbon sourcecomprising,

-   -   a) obtaining an altered bacterial strain which comprises        inactivating a chromosomal gene in a bacterial host strain,    -   b) culturing said altered bacterial host strain under suitable        conditions, and    -   c) allowing the production of an ascorbic acid intermediate from        the carbon source, wherein the production of said ascorbic acid        intermediate is enhanced compared to the production of the        ascorbic acid intermediate in the unaltered bacterial host        strain.

The ascorbic acid intermediate can be selected from the group consistingof gluconate, 2-keto-D-gluconate, 2,5-diketo-D-gluconate,2-keto-L-gulonic acid, L-iodonic acid, erthorbic acid, and tartrate. Ina particular embodiment, the intermediate is 2,5-diketo-D-gluconate. Thealtered bacterial strain can be obtained by the inactivation of a glkchromosomal gene. The altered bacterial strain can also be obtained bythe inactivation of a gntk chromosomal gene. The altered bacterialstrain can also be obtained by the inactivation of a glk and a gntkchromosomal gene. The bacterial strain can be Pantoea, morespecifically, Pantoea citrea.

In one embodiment, such host cells have a modification in apolynucleotide encoding an enzymatic activity that phosphorylatesD-glucose at its 6th carbon and/or a modification in a polynucleotideencoding an enzymatic activity that phosphorylates D-gluconate at its6th carbon. Such host cells when cultured in the presence of a carbonsource demonstrated increased yield of product as measured directlyand/or indirectly. In some embodiments of the invention, the methodscomprise host cells having a modification in a polynucleotide encodingan enzymatic activity that phosphorylates D-glucose at its 6th carbonand a modification in a polynucleotide encoding an enzymatic activitythat phosphorylates D-gluconate at its 6th carbon.

Accordingly, in one aspect, the invention provides a process forproducing an ascorbic acid intermediate in a recombinant host cellcomprising, culturing a host cell capable of producing said ascorbicacid intermediate in the presence of glucose, or a carbon source thatcan be converted to glucose by the host cell, under conditions suitablefor the production of said ascorbic acid intermediate wherein said hostcell is modified to reduce the glucose diverted to the catabolic pathwayof the organism. The modification, can comprise at least onepolynucleotide encoding an enzymatic activity that diverts such carbonsubstrate to such catabolic pathway is inactivated or deleted such thatthe level of said enzymatic activity is correspondingly affected duringsaid culturing.

In some embodiments of the present invention, the host cell furthercomprises at least one polynucleotide encoding an enzymatic activitythat phosphorylates D-gluconate at its 6th carbon wherein saidpolynucleotide has been modified such that the level of said enzymaticactivity is modified during said culturing to inactivate or reduce theamount of substrate transported across the cell membrane and/orphosphorylated for utilization within the cell's catabolic pathways. Insome embodiments, the process comprises the step of recovering saidproduct and in other embodiments, the process comprises the step ofconverting said product to a second product.

In some embodiments, the level of endogenous enzymatic activity isregulated during culturing, such as by transcriptional or translationalregulatory elements. Accordingly, in some embodiments, thepolynucleotide encoding an enzymatic activity that phosphorylatesD-glucose for transport across the cell membrane is operably linked to aregulatable promoter. In other embodiments, the polynucleotide encodingan enzymatic activity that phosphorylates D-gluconate at its 6th carbonis operably linked to a regulatable promoter. In other embodiments, theendogenous enzymatic activity is inactivated, such as by mutation in ordeletion of part or all of a polynucleotide encoding the enzymaticactivity.

The present invention encompasses production of ascorbic acidintermediates including gluconate, 2-keto-D-gluconate (2-KDG or KDG);2,5-diketo-D-gluconate (2,5DKG or DKG); 2-keto-L-gulonic acid (2KLG, orKLG); or L-idonic acid (IA). In some embodiments, the ascorbic acidintermediate is KLG and the KLG is converted to ascorbic acid. In otherembodiments, the ascorbic acid intermediate is KDG and the KDG isconverted to erythorbate.

The present invention provides recombinant host cells capable ofproducing an ASA intermediate, wherein said host cell comprises apolynucleotide encoding an enzymatic activity that phosphorylatesD-glucose at its 6th carbon, wherein said polynucleotide is modified.The invention also provides recombinant host cells further comprising apolynucleotide encoding an enzymatic activity that phosphorylatesD-gluconate at its 6th carbon, wherein said polynucleotide is modified.

In some embodiments, the host cell is a Gram positive microorganism andin other embodiments, the host cell is a Gram negative microorganism. Insome embodiments, the Gram negative host cell is an Enterobacteriaceaehost cell including Erwinia, Enterobacter, Gluconobacter, Acetobacter,Corynebacteria, Escherichia, Salmonella, Klebsiella or Pantoea.

In other embodiments, the host cell can be any bacteria that naturallyor after proper genetic modifications, is able to utilize one carbonsource to maintain certain cell functions, for example, but not limitedto, the generation of reducing power in the form of NAD, FADH₂ or NADPH,while another carbon source is converted into one or more product(s) ofcommercial interest.

In some embodiments, the enzymatic activity that phosphorylatesD-glucose at its 6^(th) carbon includes glucokinase, hexokinase, thephosphotransferase system (PTS) or any other enzyme(s) not necessarilyclassified under the 3 previous groups, but capable of such a function.For example, it is possible that an enzyme maybe classified as afructokinase because its preferred substrate is fructose, canphosphorylate D-glucose at a measurable rate. It is not uncommon thatenzymes will act on other similar substrates, especially at high levelsof substrates.

In other embodiments, the enzymatic activity that phosphorylatesD-gluconate at its 6^(th) carbon includes gluconokinase,deoxygluconokinase, hexokinase, the phosphotransferase system (PTS) orany other enzyme(s) not necessarily classified under the 4 previousgroups, but capable of such a function. For example, It is possible thatan enzyme maybe classified as a deoxyglukono-kinase because itspreferred substrate is deoxygluconate, can phosphorylate D-gluconate ata measurable rate. It is not uncommon that enzymes will act on othersimilar substrates, especially at high levels of substrates.

The present invention also provides methods for producing host cellshaving modified levels of enzymatic activities. The present inventionalso provides novel nucleic acid and amino acid sequences for enzymaticactivity that phosphorylates D-glucose at its 6th carbon and enzymaticactivity that phosphorylates D-gluconate at its 6th carbon.

The present invention also provides methods for producing host cellscapable to produce different biomolecules of industrial importancederived from glucose and/or fructose.

The present invention also provides host cells having modified levels ofenzymatic activities. The present invention also provides novel nucleicacid and amino acid sequences for enzymatic activity that phosphorylatesD-glucose at its 6th carbon and enzymatic activity that phosphorylatesD-gluconate at its 6th carbon.

The present invention also provides host cells capable to producedifferent biomolecules of industrial importance derived from glucoseand/or fructose.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides a schematic representation of some of the metabolicroutes involved in Glucose assimilation in Pantoea citrea. The enzymaticsteps affected by the genetic modifications described in the presentinvention, are indicated by an X. Boxes labeled with a T representputative transporters.

FIG. 2. Some possible catabolic routes that can be used to channelglucose into cellular metabolism. The arrows represent at least oneenzymatic step.

FIG. 3. depicts products that can be obtained from indicated commercialroutes. The majority of the carbon used to synthesize the compoundslisted on the left side, can be obtained from the catabolic pathway orTCA cycle. On the contrary, the compounds on the right, derive most ofits carbon from the pentose pathway and/or from the oxidation of glucoseinto keto-acids.

FIG. 4 depicts a nucleic acid (SEQ ID NO:1) sequence for a Pantoeacitrea glucokinase (glk).

FIG. 5 depicts an amino acid (SEQ ID NO:2) sequence for a Pantoea citreaglucokinase (Glk).

FIG. 6 depicts a nucleic acid (SEQ ID NO:3) sequence for a Pantoeacitrea gluconokinase (gntk)

FIG. 7 depicts an amino acid (SEQ ID NO:4) sequence for a Pantoea citreagluconokinase (Gntk).

FIG. 8 depicts amino acid (SEQ ID NO: 8-13) for the genes glk 30, glk31, gnt 1, gnt 2, pcgnt 3 and pcgnt 4.

FIG. 9 depicts D-glucose, D-gluconate and some of their derivatives. Thestandard numbering of the carbons on glucose is indicated by the numbers1 and 6. 2-KDG=2-keto-D-gluconate; 2,5-DKG=2,5-diketogluconate;2KLG=2-keto-L-gulonate.

FIG. 10 depicts general strategy used to interrupt the gluconate kinasegene from P. citrea.

FIG. 11 depicts the oxidative pathway for the production of ascorbicacid. E1 stands for glucose dehydrogenase; E2 stands for gluconic aciddehydrogenase; E3 stands for 2-keto-Dgluconic acid dehydrogenase; and E4stands for 2,5-diketo-D-gluconic acid reductase.

FIG. 12 depicts the net reactions during the fermentation of host cellscapable of producing ascorbic acid intermediates.

FIG. 13 depicts carbon evolution rate (CER) and oxygen uptake rate (OUR)of a fermentation of a wild-type organism after exposure to glucose.

FIG. 14 depicts the CER and OUR of a fermentation with a single delete(glucokinase).

FIG. 15 depicts the CER and OUR of a fermentation with a single delete(gluconokinase).

FIG. 16 depicts the CER and OUR of a fermentation with a host cellhaving both glucokinase and gluconokinase deleted.

FIG. 17 is a schematic illustrating the interrelationships of variousmetabolic pathways (including the glycolytic pathway, TCA cycle, andpentose pathway) and the oxidative pathways. Glk=glucokinase;Gntk=gluconokinase; IdnO=Idonate reductase; IdnD=Idonate dehydrogenase;TKT=transketolase; TAL=transaldolase, 2KR=2-keto reductase;2,5DKGR=2,5-diketogluconate reductase.

FIG. 18 is a schematic illustrating the interrelationships of variouscentral metabolic pathways and the modifications which would increasethe production of gluconate. The X indicate the enzymatic pathways thatwould be modified to effect the desired increase in gluconateproduction.

FIG. 19 is a schematic illustrating the interrelationships of variouscentral metabolic pathways and the modifications which would increasethe production of erythorbic acid. The X indicate the enzymatic pathwaysthat would be modified to effect the desired increase in erythorbic acidproduction.

FIG. 20 is a schematic illustrating the interrelationships of variouscentral metabolic pathways and the modifications which would increasethe production of ribose. The X indicate the enzymatic or transportpathways that would be modified to effect the desired increase in2,5-diketogluconate production.

FIG. 21 is a schematic illustrating the interrelationships of variouscentral metabolic pathways and the modifications which would increasethe production of tartrate. The X indicate the enzymatic steps thatwould be modified to effect the desired increase in ribose production.IdnO=5-keto-D-gluconate 5-reductase; IdnD=I-Idonate 5-dehydrogenase.

FIG. 22 is a schematic illustrating the pathway of dihydroxyacetonephosphate (DHAP) being converted to glycerol.

FIG. 23 depicts the DNA Sequence of primers used to amplify by PCR the2.9 kb DNA fragment that contains the glpK gene as described in Example7.

FIG. 24 describes the DNA sequence of the structural gene of theglycerol kinase from P. citrea as described in Example 7. The sequenceof the Hpal site used to interrupt the gene is underlined.

FIG. 25 depicts the protein sequence of the glycerol kinase from P.citrea as described in Example 7.

DETAILED DESCRIPTION

The invention provides methods and host cells for the production ofascorbic acid intermediates. Recombinant host cells have beenconstructed which produce ascorbic acid intermediates and which aregenetically engineered to reduce the carbon substrate diverted tocatabolic pathways. In one embodiment, the recombinant host cellsproduce modified levels of enzymatic activity that transports into thecell for use in the catabolic pathways, e.g., those that phosphorylateD-glucose at its 6th carbon and/or enzymatic activity thatphosphorylates D-gluconate at its 6th carbon. When these recombinantcells are cultured in the presence of glucose or a carbon source whichcan be converted to glucose by the host cell, intracellular metabolismof the glucose and/or gluconate is reduced.

In some embodiments, the host cell is a Pantoea citrea cell which hasbeen genetically engineered to produce a product, such as an ascorbicacid (“ASA”) intermediate. In these embodiments, a particular advantageprovided by the invention is the ability to make use of fermentationprocesses for the production of the ASA intermediate.

Another advantage provided by the invention is the uncoupling of theextracellular oxidation of a substrate from the metabolic pathways thatuse those oxidation products.

Another advantage provided by the invention is the increased efficiencyin the production of ascorbic acid intermediates as compared towild-type bioconversion as measured directly by increased production ofthe desired product or indirectly as measured by oxygen consumption orCO₂ off gas (CO₂ production).

A further advantage provided by the invention is the ability of the hostcell to utilize two different carbon sources simultaneously for theproduction of ascorbic acid intermediates.

General Techniques

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology (includingrecombinant techniques), microbiology, cell biology, and biochemistry,which are within the skill of the art. Such techniques are explainedfully in the literature, such as, Molecular Cloning: A LaboratoryManual, second edition (Sambrook et al., 1989); Current Protocols inMolecular Biology (F. M. Ausubel et al., eds., 1987 and annual updates);Oligonucleotide Synthesis (M. J. Gait, ed., 1984); and PCR: ThePolymerase Chain Reaction, (Mullis et al., eds., 1994). Manual ofIndustrial Microbiology and Biotechnology, Second Edition (A. L. Demain,et al., eds. 1999)

DEFINITIONS

As used herein, “oxidative pathway of a host cell” means that the hostcell comprises at least one oxidative enzyme that oxidizes a carbonsource, such as, D-glucose and/or its metabolites. A “membrane” or“membrane bound” glucose oxidative pathway in a host cells refers to ahost cell that oxidizes a carbon source such as, D-glucose and/or itsmetabolites, via at least one membrane bound oxidative enzymaticactivity. In some embodiments, an oxidative pathway in a host cellcomprises one enzymatic activity. In other embodiments, an oxidativepathway in a host cell comprises two or more enzymatic activities.

As used herein, “catabolic pathway of a host cell” means that the hostcell comprises at least one enzymatic activity that generates ATP orNADPH, for example, by phosphorylating a carbon source, such asD-glucose and/or its metabolites. An “intracellular” catabolic pathwayin a host cell means that the host cell comprises at least one suchenzymatic activity in the host cell cytosol. In some embodiments, acatabolic pathway in a host cell comprises one enzymatic activity. Inother embodiments, a catabolic pathway in a host cell comprises two ormore enzymatic activities. Catabolic pathways include, but are notlimited to glycolysis, the pentose pathway and the TCA pathway (see FIG.17).

As used herein, the phrase “enzymatic transport system” refers to anenzymatic activity that consumes energy (ATP) and transports the carbonsubstrate across the cell membrane, usually by adding a phosphate to the6th carbon of D-glucose and includes the enzymatic activitiesglucokinase (E.C.-2.7.1.2) and phosphotransferase system (PTS)(E.C.-2.7.1.69).

As used herein, the phrase “enzymatic activity which phosphorylatesD-gluconate at its 6th carbon” refers to an enzymatic activity thatphosphorylates D-gluconate at its 6th carbon and includes the enzymaticactivity gluconokinase (E.C.-2.7.1.12).

As used herein, the phrase “enzymatic activity which phosphorylatesD-glucose at its 6th carbon” refers to an enzymatic activity that adds aphosphate to the 6th carbon of D-glucose and includes the enzymaticactivities glucokinase (E.C.-2.7.1.2); and phosphotransferase system(PTS) (E.C.-2.7.1.69).

As used herein, the phrase “enzymatic activity which phosphorylatesD-gluconate at its 6th carbon” refers to an enzymatic activity thatphosphorylates D-gluconate at its 6th carbon and includes the enzymaticactivity gluconokinase (E.C.-2.7.1.12).

As used herein, “modifying” the levels of an enzymatic activity producedby a host cell or “modified levels” of an enzymatic activity of a hostcell refers to controlling the levels of enzymatic activity producedduring culturing, such that the levels are increased or decreased asdesired. The desired change in the levels of enzymatic activity may begenetically engineered to take place in one or both enzymatic activitieseither simultaneously or sequentially, in any order. In order to controlthe levels of enzymatic activity, the host cell is geneticallyengineering such that nucleic acid encoding the enzymatic activity istranscriptionally or translationally controlled.

As used herein, the term “modified” when referring to nucleic acid orpolynucleotide means that the nucleic acid has been altered in some wayas compared to wild type nucleic acid, such as by mutation in; deletionof part or all of the nucleic acid; or by being operably linked to atranscriptional control region. As used herein the term “mutation” whenreferring to a nucleic acid refers to any alteration in a nucleic acidsuch that the product of that nucleic acid is partially or totallyinactivated or eliminated. Examples of mutations include but are notlimited to point mutations, frame shift mutations and deletions of partor all of a gene encoding an enzymatic activity, such as an enzymaticactivity that transports the substrate across the cell membrane, e.g.,phosphorylates D-glucose at its 6th carbon or an enzymatic activity thatphosphorylates D-gluconate at its 6th carbon.

An “altered bacterial strain” according to the invention is agenetically engineered bacterial microorganism having an enhanced levelof production over the level of production of the same end-product in acorresponding unaltered bacterial host strain grown under essentiallythe same growth conditions. An “unaltered bacterial strain” or host is abacterial microorganism wherein the coding sequence of the divertingenzymatic pathway is not inactivated and remains enzymatically active.The enhanced level of production results from the inactivation of one ormore chromosomal genes. In a first embodiment the enhanced level ofexpression results from the deletion of one or more chromosomal genes.In a second embodiment the enhanced level of expression results from theinsertional inactivation of one or more chromosomal genes. Preferablythe inactivated genes are selected from those encoding the enzymes whoseinactivity is desired as described elsewhere in this application. Forexample, in one embodiment one or more chromosomal genes selected fromthe group consisting of glk, and gntk.

In certain embodiments, the altered Bacillus strain may embody twoinactivated genes, three inactivated genes, four inactivated genes, fiveinactivated genes, six inactivated genes or more. The inactivated genesmay be contiguous to one another or may be located in separate regionsof the Bacillus chromosome. An inactivated chromosomal gene may have anecessary function under certain conditions, but the gene is notnecessary for Bacillus strain viability under laboratory conditions.Preferred laboratory conditions include but are not limited toconditions such as growth in a fermentator, in a shake plate, in platemedia or the like.

As used herein, the term “inactivation” or “inactivating” when referringto an enzymatic activity means that the activity has been eliminated byany means including a mutation in or deletion of part or all of thenucleic acid encoding the enzymatic activity. The term “inactivation” or“inactivating” includes any method that prevents the functionalexpression of one or more of the desired chromosomal genes, wherein thegene or gene product is unable to exert its known function. The desiredchromosomal genes will depend upon the enzymatic activity that isintended to be inactivated. For example the inactivation of glucokinaseand/or gluconokinase activity can be effected by inactivating the glkand/or gntk chromosomal genes. Inactivation may include such methods asdeletions, mutations, interruptions or insertions in the nucleic acidgene sequence. In one embodiment, the expression product of aninactivated gene may be a truncated protein as long as the protein has achange in its biological activity. The change in biological activitycould be an altered activity, but preferably is loss of biologicalactivity. In an altered bacterial strain according to the invention, theinactivation of the one or more genes will preferably be a stable andnon-reverting inactivation.

In a preferred embodiment, preferably a gene is deleted by homologousrecombination. For example, as shown in FIG. 9, when glk is the gene tobe deleted, a chloramphenicol resistance gene is cloned into a uniquerestriction site found in the glucokinase gene. The Cm^(R) gene isinserted into the structural coding region of the gene at the Pst Isite. Modification is then transferred to the chromosome of a P. citreaglkA- by homologous recombination using a non-replication R6K vector.The Cm^(R) gene is subsequently removed from the glk coding regionleaving an interrupting spacer in the coding region, inactivating thecoding region. In another embodiment, the Cm^(R) gene is inserted intothe coding region in exchange for portions of the coding region.Subsequent removal of the Cm^(R) gene without concomitant reinsertion ofthe exchanged out portion of the coding region results in an effectivedeletion of a portion of the coding region, inactivating such region.

A deletion of a gene as used herein may include deletion of the entirecoding sequence, deletion of part of the coding sequence, or deletion ofthe coding sequence including flanking regions. The deletion may bepartial as long as the sequences left in the chromosome are too shortfor biological activity of the gene. The flanking regions of the codingsequence may include from about 1 bp to about 500 bp at the 5′ and 3′ends. The flanking region may be larger than 500 bp but will preferablynot include other genes in the region which may be inactivated ordeleted according to the invention. The end result is that the deletedgene is effectively non-functional.

In another preferred embodiment, inactivation is by insertion. Forexample when glk is the gene to be inactivated, a DNA construct willcomprise an incoming sequence having the glk gene interrupted by aselective marker. The selective marker will be flanked on each side bysections of the glk coding sequence. The DNA construct aligns withessentially identical sequences of the glk gene in the host chromosomeand in a double crossover event the glk gene is inactivated by theinsertion of the selective marker.

In another embodiment, activation is by insertion in a single crossoverevent with a plasmid as the vector. For example, a glk chromosomal geneis aligned with a plasmid comprising the gene or part of the gene codingsequence and a selective marker. The selective marker may be locatedwithin the gene coding sequence or on a part of the plasmid separatefrom the gene. The vector is integrated into the Bacillus chromosome,and the gene is inactivated by the insertion of the vector in the codingsequence.

Inactivation may also occur by a mutation of the gene. Methods ofmutating genes are well known in the art and include but are not limitedto chemical mutagenesis, site-directed mutation, generation of randommutations, and gapped-duplex approaches. (U.S. Pat. No. 4,760,025;Moring et al., Biotech. 2:646 (1984); and Kramer et al., Nucleic AcidsRes. 12:9441 (1984)).

Inactivation may also occur by applying the above described inactivationmethods to the respective promoter regions of the desired genomicregion.

“Under transcriptional control” or “transcriptionally controlled” areterms well understood in the art that indicate that transcription of apolynucleotide sequence, usually a DNA sequence, depends on its beingoperably (operatively) linked to an element which contributes to theinitiation of, or promotes, transcription. “Operably linked” refers to ajuxtaposition wherein the elements are in an arrangement allowing themto function.

As used herein, the term “regulatable promoter” refers to a promoterelement which activity or function can be modulated. This modulation canbe accomplished in many different ways, most commonly by the interactionof protein(s) that interfere or increase the ability of the RNApolymerase enzyme to initiate transcription.

“Under translational control” is a term well understood in the art thatindicates a regulatory process that occurs after the messenger RNA hasbeen formed.

As used herein, the term “batch” describes a batch cell culture to whichsubstrate, in either solid or concentrated liquid form, is addedinitially at the start of the run. A batch culture is initiated byinoculating cells to the medium, but, in contrast to a fed-batchculture, there is no subsequent inflow of nutrients, such as by way of aconcentrated nutrient feed. In contrast to a continuous culture, in abatch cell culture, there is no systematic addition or systematicremoval of culture fluid or cells from a culture. There is no ability tosubsequently add various analytes to the culture medium, since theconcentrations of nutrients and metabolites in culture medium aredependent upon the initial concentrations within the batch and thesubsequent alteration of the composition of the nutrient feed due to theact of fermentation.

As used herein, the term “fed-batch” describes a batch cell culture towhich substrate, in either solid or concentrated liquid form, is addedeither periodically or continuously during the run. Just as in a batchculture, a fed-batch culture is initiated by inoculating cells to themedium, but, in contrast to a batch culture, there is a subsequentinflow of nutrients, such as by way of a concentrated nutrient feed. Incontrast to a continuous culture there is no systematic removal ofculture fluid or cells from a fed-batch culture is advantageous inapplications that involve monitoring and manipulating the levels ofvarious analytes in the culture medium, since the concentrations ofnutrients and metabolites in culture medium can be readily controlled oraffected by altering the composition of the nutrient feed. The nutrientfeed delivered to a fed-batch culture is typically a concentratednutrient solution containing an energy source, e.g., carbohydrates;optionally, the concentrated nutrient solution delivered to a fed-batchculture can contain amino acids, lipid precursors and/or salts. In afed-batch culture, this nutrient feed is typically rather concentratedto minimize the increase in culture volume while supplying sufficientnutrients for continued cell growth.

The term “continuous cell culture” or, simply, “continuous culture” isused herein to describe a culture characterized by both a continuousinflow of a liquid nutrient feed and a continuous liquid outflow. Thenutrient feed may, but need not, be a concentrated nutrient feed.Continuously supplying a nutrient solution at about the same rate thatcells are washed out of the reactor by spent medium allows maintenanceof a culture in a condition of stable multiplication and growth.

In a type of bioreactor known as a chemostat, the cell culture iscontinuously fed fresh nutrient medium, and spent medium, cells andexcreted cell product are continuously drawn off. Alternatively, acontinuous culture may constitute a “perfusion culture,” in which casethe liquid outflow contains culture medium that is substantially free ofcells, or substantially lower cell concentration than that in thebioreactor. In a perfusion culture, cells can be retained by, forexample, filtration, centrifugation, or sedimentation.

“Culturing” as used herein refers to fermentative bioconversion of acarbon substrate to the desired end-product within a reactor vessel.Bioconversion as used herein refers to the use of contacting amicroorganism with the carbon substrate to convert the carbon substrateto the desired end-product.

As used herein, “Oxygen Uptake Rate or “OUR” refers to the determinationof the specific consumption of oxygen within the reactor vessel. Oxygenconsumption can be determined using various on-line measurements. In oneexample, the OUR (mmol/(liter*hour)) is determined by the followingformula: ((Airflow (standing liters per minute)/Fermentation weight(weight of the fermentation broth in kilograms))×supply O₂×brothdensity×(a constant to correct for airflow calibration at 21.1 C insteadof standard 20.0 C)) minus ([airflow/fermentation weight]×[offgasO₂/offgas N₂]×supply N₂×broth density×constant).

As used herein, “carbon evolution rate or “CER” refers to thedetermination of how much CO₂ is produced within the reactor vesselduring fermentation. Usually, since no CO₂ is initially or subsequentlyprovided to the reaction vessel, any CO₂ is assumed to be produced bythe fermentation process occurring within the reaction vessel. “Off-gasCO₂” refers to the amount of CO₂ measured within the reactor vessel,usually by mass spectroscopic methods known in the art.

As used herein, “yield” refers to the amount of product/the amount ofsubstrate. The yield can be expressed as a weight % (productgm/substrate gm) or as moles of product/moles of substrate. For example,the amount of the substrate, e.g., glucose can be determined by the feedrate and the concentration of the added glucose. The amount of productspresent can be determined by various spectrophotometric or analyticmethodologies. One such methodology is high performance liquidchromatography (HPLC). An increased yield refers to an increased yieldas compared to the yield of a conversion using the wild-type organism,for example an increase of 10%, 20% or 30% over the yield of thewild-type.

The phrase “productive enzyme” as used herein refers to an enzyme orenzyme system which can catalyze conversion of a substrate to a desiredproduct. Productive enzymes include, but are not limited to oxidativeenzymes and reducing enzymes.

The phrase “oxidative enzyme” as used herein refers to an enzyme orenzyme system which can catalyze conversion of a substrate of a givenoxidation state to a product of a higher oxidation state than substrate.The phrase “reducing enzyme” refers to an enzyme or enzyme system whichcan catalyze conversion of a substrate of a given oxidation state to aproduct of a lower oxidation state than substrate. In one illustrativeexample disclosed herein, oxidative enzymes associated with thebiocatalysis of D-glucose or its metabolites in a Pantoea cell which hasbeen engineered to produce ASA intermediates, include among othersD-glucose dehydrogenase, D-gluconate dehydrogenase and2-keto-D-gluconate dehydrogenase. In another illustrative embodimentdisclosed herein, reducing enzymes associated with the biocatalysis ofD-glucose or its metabolites in a Pantoea cell which has been engineeredto produce ASA intermediates, as described herein, include among others2,5-diketo-D-gluconate reductase, 2-keto reductase and 5-keto reductase.Such enzymes include those produced naturally by the host strain orthose introduced via recombinant means.

As used herein, the term “carbon source” encompasses suitable carbonsources ordinarily used by microorganisms, such as 6 carbon sugars,including but not limited to glucose, gulose, sorbose, fructose, idose,galactose and mannose all in either D or L form, or a combination of 6carbon sugars, such as glucose and fructose, and/or 6 carbon sugar acidsincluding but not limited to 2-keto-L-gulonic acid, idonic acid,gluconic acid, 6-phosphogluconate, 2-keto-D-gluconic acid,5-keto-D-gluconic acid, 2-ketogluconatephosphate, 2,5-diketo-L-gulonicacid, 2,3-L-diketogulonic acid, dehydroascorbic acid, erythrorbic acidand D-mannonic acid or the enzymatic derivatives of such.

The following abbreviations apply as used herein to D-glucose or glucose(G); D-gluconate or gluconate (GA); 2-keto-D-gluconate (2 KDG);2,5-diketo-D-gluconate (2,5DKG or DKG); 2-keto-L-gulonic acid (2KLG, orKLG); L-idonic acid (IA); erythorbic acid (EA); ascorbic acid (ASA);glucose dehydrogenase (GDH); gluconic acid dehydrogenase (GADH);2,5-diketo-D-gluconate reductase (DKGR); 2-keto-D-gluconate reductase(KDGDH); D-ribose (R); 2-keto reductase (2KR or KR); and 5-ketoreductase (5KR or KR).

“Allowing the production of an ascorbic acid intermediate from thecarbon source, wherein the production of said ascorbic acid intermediateis enhanced compared to the production of the ascorbic acid intermediatein the unaltered bacterial host strain” means contacting the substrate,e.g. carbon source, with the altered bacterial strain to produce thedesired end-product. The inventors discovered that by altering certainenzymatic activities by inactivating genomic expression, themicroorganism demonstrated enhanced end-product production.

“Desired end-product” as used herein refers to the desired compound towhich the carbon substrate is bioconverted into. The desired end-productmay be the actual compound sought or an intermediate along anotherpathway. Exemplary desired end-products are listed on the right side ofFIG. 3.

As used herein, the term bacteria refers to any group of microscopicorganisms that are prokaryotic, i.e., that lack a membrane-bound nucleusand organelles. All bacteria are surrounded by a lipid membrane thatregulates the flow of materials in and out of the cell. A rigid cellwall completely surrounds the bacterium and lies outside the membrane.There are many different types of bacteria, some of which include, andare not limited to those strains within the families ofEnterobacteriaceae, Bacillus, Streptomyces, Pseudomonas, and Erwinia.

As used herein, the family “Enterobacteriaceae” refers to bacterialstrains having the general characteristics of being Gram negative andbeing facultatively anaerobic, including the genus contained withinEscherichia coli, Shigella, Edwardsiella; Salmonelle; Citrobacter,Klebsiella, Enterobacter, Serratia, Proteus, Morgarella, Providencia,and yersinia. For the production of ASA intermediates, preferredEnterobacteriaceae strains are those that are able to produce2,5-diketo-D-gluconic acid from D-glucose or carbon sources which can beconverted to D-glucose by the strain. Included in the family ofEnterobacteriaceae which are able to produce 2,5-diketo-D-gluconic acidfrom D-glucose solutions are the genus Erwinia, Enterobacter,Gluconobacter and Pantoea, for example. Intermediates in the microbialpathway from carbon source to ASA, include but are not limited to GA,KDG, DKG, DKG, KLG, IA and EA. In the present invention, a preferredEnterobacteriaceae fermentation strain for the production of ASAintermediates is a Pantoea species and in particular, Pantoea citrea.Other Enterobacteriaceae strains that produce ASA intermediates include,but are not limited to, E. coli and Gluconobacter.

As used herein the family “Bacillus” refers to rod-shaped bacterialstrains having the general characteristics of being gram positive,capable of producing spores under certain environmental conditions.

As used herein, the term “recombinant” refers to a host cell that has amodification of its genome, e.g., as by the additional of nucleic acidnot naturally occurring in the organism or by a modification of nucleicacid naturally occurring in the host cell and includes host cells havingadditional copies of endogenous nucleic acid introduced via recombinantmeans. The term “heterologous” as used herein refers to nucleic acid oramino acid sequences not naturally occurring in the host cell. As usedherein, the term “endogenous” refers to a nucleic acid naturallyoccurring in the host.

The terms “isolated” or “purified” as used herein refer to an enzyme, ornucleic acid or protein or peptide or co-factor that is removed from atleast one component with which it is naturally associated. In thepresent invention, an isolated nucleic acid can include a vectorcomprising the nucleic acid.

It is well understood in the art that the acidic derivatives ofsaccharides, may exist in a variety of ionization states depending upontheir surrounding media, if in solution, or out of solution from whichthey are prepared if in solid form. The use of a term, such as, forexample, idonic acid, to designate such molecules is intended to includeall ionization states of the organic molecule referred to. Thus, forexample, “idonic acid”, its cyclized form “idonolactone”, and “idonate”refer to the same organic moiety, and are not intended to specifyparticular ionization states or chemical forms.

As used herein, the term “vector” refers to a polynucleotide constructdesigned for transduction/transfection of one or more cell typesincluding for example, “cloning vectors” which are designed forisolation, propagation and replication of inserted nucleotides or“expression vectors” which are designed for expression of a nucleotidesequence in a host cell, such as a Pantoea citrea or E. coli host cell.

The terms “polynucleotide” and “nucleic acid”, used interchangeablyherein, refer to a polymeric form of nucleotides of any length, eitherribonucleotides or deoxyribonucleotides. These terms include a single-,double- or triple-stranded DNA, genomic DNA, cDNA, RNA, DNA-RNA hybrid,or a polymer comprising purine and pyrimidine bases, or other natural,chemically, biochemically modified, non-natural or derivatizednucleotide bases. The backbone of the polynucleotide can comprise sugarsand phosphate groups (as may typically be found in RNA or DNA), ormodified or substituted sugar or phosphate groups. Alternatively, thebackbone of the polynucleotide can comprise a polymer of syntheticsubunits such as phosphoramidates and thus can be a oligodeoxynucleosidephosphoramidate (P—NH2) or a mixed phosphoramidate-phosphodiesteroligomer. Peyrottes et al. (1996) Nucleic Acids Res. 24: 1841-8;Chaturvedi et al. (1996) Nucleic Acids Res. 24: 2318-23; Schultz et al.(1996) Nucleic Acids Res. 24: 2966-73. A phosphorothioate linkage can beused in place of a phosphodiester linkage. Braun et al. (1988) J.Immunol. 141: 2084-9; Latimer et al. (1995) Molec. Immunol. 32:1057-1064. In addition, a double-stranded polynucleotide can be obtainedfrom the single stranded polynucleotide product of chemical synthesiseither by synthesizing the complementary strand and annealing thestrands under appropriate conditions, or by synthesizing thecomplementary strand de novo using a DNA polymerase with an appropriateprimer. Reference to a polynucleotide sequence (such as referring to aSEQ ID NO) also includes the complement sequence.

The following are non-limiting examples of polynucleotides: a gene orgene fragment, exons, introns, mRNA, tRNA, rRNA, ribozymes, cDNA,recombinant polynucleotides, branched polynucleotides, plasmids,vectors, isolated DNA of any sequence, isolated RNA of any sequence,nucleic acid probes, and primers. A polynucleotide may comprise modifiednucleotides, such as methylated nucleotides and nucleotide analogs,uracyl, other sugars and linking groups such as fluororibose andthioate, and nucleotide branches. The sequence of nucleotides may beinterrupted by non-nucleotide components. A polynucleotide may befurther modified after polymerization, such as by conjugation with alabeling component. Other types of modifications included in thisdefinition are caps, substitution of one or more of the naturallyoccurring nucleotides with an analog, and introduction of means forattaching the polynucleotide to proteins, metal ions, labelingcomponents, other polynucleotides, or a solid support. Preferably, thepolynucleotide is DNA. As used herein, “DNA” includes not only bases A,T, C, and G, but also includes any of their analogs or modified forms ofthese bases, such as methylated nucleotides, internucleotidemodifications such as uncharged linkages and thioates, use of sugaranalogs, and modified and/or alternative backbone structures, such aspolyamides.

A polynucleotide or polynucleotide region has a certain percentage (forexample, 80%, 85%, 90%, 95%, 97% or 99%) of “sequence identity” toanother sequence means that, when aligned, that percentage of bases arethe same in comparing the two sequences. This alignment and the percenthomology or sequence identity can be determined using software programsknown in the art, for example those described in Current Protocols inMolecular Biology (F. M. Ausubel et al., eds., 1987) Supplement 30,section 7.7.18. A preferred alignment program is ALIGN Plus (Scientificand Educational Software, Pennsylvania), preferably using defaultparameters, which are as follows: mismatch=2; open gap=0; extend gap=2.

A polynucleotide sequence that is “depicted in” a SEQ ID NO means thatthe sequence is present as an identical contiguous sequence in the SEQID NO. The term encompasses portions, or regions of the SEQ ID NO aswell as the entire sequence contained within the SEQ ID NO.

“Expression” includes transcription and/or translation.

As used herein, the term “comprising” and its cognates are used in theirinclusive sense; that is, equivalent to the term “including” and itscorresponding cognates.

“A,” “an” and “the” include plural references unless the context clearlydictates otherwise.

Enzymatic Activities

Esters of phosphoric acid are encountered with trioses, tetroses,pentoses, hexoses and heptoses. The phosphorylation of all sugars is theinitial step in their metabolism. Thus glucose can be phosphorylated toglucose 6-phospahte. All cells that can metabolize glucose contain someform of a hexokinase which catalyze the reaction

Mg²⁺

Glucose+ATP→glucose 6-phosphate+ADP

FIG. 9 depicts D-glucose and illustrates the “6th carbon”. Exemplaryhexokinases include hexokinase (Frohlich, et al., 1985, Gene 36:105-111)and glucokinase (Fukuda, et al., 1983, J. Bacteriol. 156:922-925). TheDNA sequence of the glucokinase structural gene from P. citrea is shownin FIG. 4. The recogition site for the restriction enzymes NcoI (CCATGG)and SnaBI (TACGTA) are highlighted. FIG. 5 depicts the protein sequenceof the glucokinase gene from P. citrea. Most hexokinases are somewhatnonspecific, showing some ability to catalyze formation of 6-phosphateesters of mannose, fructose, and galactose. In addition, other hexosederivatives may also be phosphorylated by a hexokinase. Gluconate (FIG.3), for example, may also be phosphorylated by a kinase, specificallygluconokinase (citation). The sequence for the gluconokinase structuralgene from P. citrea is depicted in FIG. 6. The recognition site for therestriction enzyme Pst I (CTGCAG) is highlighted. The protein sequencefor the gluconokinase gene from P. citrea is depicted in FIG. 7 (SEQ IDNO 4). The some of the genes for glucokinase and gluconokinase (glk,gntk, etc.) are shown in FIG. 8.

FIG. 17 shows the interrelationships between the catabolic pathway andthe oxidative pathway. Glucose can enter the catabolic pathway throughthe glycolytic pathway by the phosphorylation of glucose toglucose-6-phosphate by glucokinase (Glk); and through the pentosepathway by the phosphorylation of gluconate to gluconate-6-phosphate byglucono kinase (Gntk). Inactivation or modifying the levels ofglucokinase and gluconokinase by modifying the nucleic acid orpolypeptide encoding the same (glk and/or gntk), results in theincreased yield of the desired product, e.g. an ascorbic acidintermediate. Genetic modifications are used to eliminate thecommunication between the catabolic functions and the enzymaticreactions that are required to synthesize a desired product. Whileglucokinase and gluconokinase are modified in one embodiment, theinventors contemplate that other enzymatic steps could be modified toachieve the same uncoupling oxidative, catabolic pathway uncoupling.

In another embodiment, the catabolic pathway is uncoupled from theproductive pathway to increase the production of 5-KDG and/or tartrate.As shown in FIG. 21, glucose can enter the catabolic pathway through theglycolytic pathway, for example through glucose-6-phosphate, the pentosepathway through gluconate-6-phosphate, and other ascorbic acidby-products, such as idonate and 2-KLG. Inactivation or modifying thelevels of glucokinase, gluconokinase, 2,5-DKG reductase,5-keto-D-gluconate 5-reductase (idnO) and Idonate 5-dehydrogenase(idnD), by modifying the nucleic acid or polypeptide encoding the same,results in the increased yield of the desired product, e.g. 5-DKG and/ortartrate.

In another embodiment, the catabolic pathway is uncoupled from theproductive pathway to increase the production of gluconate. As shown inFIG. 18 glucose can enter the catabolic pathway through the glycolyticpathway, for example through glucose-6-phosphate and the pentosepathway, through gluconate-6-phosphate. Inactivation or modifying thelevels of glucokinase, gluconokinase, and glyceraldhehyde hydrogenase,by modifying the nucleic acid or polypeptide encoding the same, resultsin the increased yield of the desired product, e.g. gluconate.

In another embodiment, the catabolic pathway is uncoupled from theproductive pathway to increase the production of erythorbic acid. Asshown in FIG. 19 glucose can enter the catabolic pathway through theglycolytic pathway, for example through glucose-6-phosphate; the pentosepathway, through gluconate-6-phosphate; and by an enzymatic transportsystem transporting 2-KDG and 2,5-KDG into the cytoplasm. Inactivationor modifying the levels of glucokinase, gluconokinase, and 2-KDGhydrogenase; and the transport system of 2-KDG into the cytoplasm, bymodifying the nucleic acid or polypeptide encoding the same, results inthe increased yield of the desired product, e.g. erythoric acid.

In another embodiment, the catabolic pathway is uncoupled from theproductive pathway to increase the production of 2,5-DKG. As shown inFIG. 20, glucose can enter the catabolic pathway through the glycolyticpathway, for example through glucose-6-phosphate; the pentose pathway,through gluconate-6-phosphate; and by an enzymatic transport systemtransporting 2-KDG and 2,5-KDG into the cytoplasm. Inactivation ormodifying the levels of glucokinase, gluconokinase and 2-KDGhydrogenase; and the transport system of 2-KDG into the cytoplasm bymodifying the nucleic acid or polypeptide encoding the same, results inthe increased yield of the desired product, e.g. 2,5-DKG.

The availability of recombinant techniques to effect expression ofenzymes in foreign hosts permits the achievement of the aspect of theinvention which envisions production of an ascorbic acid intermediatewith a reduced amount of carbon substrate diverted to catabolic pathwaysfrom a readily available carbon substrate. This method has considerableadvantage over presently used methods in characterized by a reduction inthe amount of substrate converted to the catabolic pathway and thusunavailable for conversion to the desired oxidative end-product, e.g.,an ascorbic acid intermediate. This results in increased fermentativeefficiency and increased yield over fermentations with wild typeorganisms. Certain wild type organisms may produce ascorbic acidintermediates, e.g., 2-KLG, however the level produced may not besufficient to be economically practical. It has been observed that wildtype Pantoea citrea has its own cytoplasmic glucokinase andgluconokinase enabling the organism to convert glucose to phosphorylatedderivatives for use in its central metabolic pathways and the productionof which, necessarily consume energy, ATP and causes that more carbongoes to non-2-KLG producing pathways. Under the same controlledconditions and using the method of this invention, described below, intwo interruption plasmid described elsewhere in this application theglucokinase and gluconokinase genes can be deleted from the P. citreagenome, enabling the modified P. citrea to produce increased DKG fromglucose at a a level increased over the wild-type, e.g. level of 63%yield to about 97-98% yield. [see Example 6].

One approach to producing a single organism conversion that is includedin this invention comprises construction of an expression vector for thedeleted kinase as outlined above, and transfer of this vector by any ofthe gene transfer methods mentioned above such as transformation,transduction or conjugation, into cells which are capable of the initialconversion of ordinary metabolites into the 2,5-DKG substrate for thisenzyme. As outlined in the example below, this gene transfer results ina organism that is characterized by an increased yield of ascorbic acidintermediates. The details of the vector construction, gene transfer,and use of the resultant organism are described in the specification.

An alternative approach is to clone the genes encoding the enzymes knownto effect the conversion of glucose or other ordinary metabolite to2,5-DKG from the organisms known to contain them, to constructexpression vectors containing these cloned gene sequences, and totransfer such vectors to cells which normally produce the 2,5-DKGreductase. Examples of the enzymes effecting the conversion of anordinary metabolite to 2,5-DKG are D-glucose dehydrogenase (Adachi, O.et al., Agric. Biol. Chem., 44(2):301-308 [1980] Ameyama, M. et al.,Agric. Biol. Chem. 45[4]:851-861 [1981]), D-gluconate dehydrogenase(McIntire, W. et al., Biochem. J., 231:651-654 [1985]; Shinagawa, E. etal., Agric. Biol. Chem. 40[3]:475-483 [1976]; Shinagawa, E. et al.,Agric. Biol. Chem. 42[5]:1055-1057 [1978]), 5-keto-D-gluconatedehydrogenase and 2-keto-D-gluconate dehydrogenase (Shinagawa, E. etal., Agric. Biol. Chem., 45(5):1079-1085 [1981]). A third approach is totransfer to a neutral host the entire sequence of enzymes comprising theconversion of an ordinary metabolite to 2-KLG. This last approach offersthe advantage of choice of host organism almost at will, for whateverdesirable growth characteristics and nutritional requirements it mayhave. Thus, the use as host cells of organisms which have the heritageof a reasonable history of experience in their culture and growth, suchas E. coli and Bacillus, confers the advantage of uniformity with otherprocedures involving bacterial production of enzymes or substrates.

Once the organism capable of carrying out the conversion has beencreated, the process of the invention may be carried out in a variety ofways depending on the nature of the construction of the expressionvectors for the recombinant enzymes and upon the growth characteristicsof the host. Typically, the host organism will be grown under conditionswhich are favorable to production of large quantities of cells. When alarge number of cells has accumulated, the promoter(s) supplied with therecombinant gene sequences either become or are already active,permitting the transcription and translation of the coding sequences.Upon suitable expression of these genes, and hence the presence of thedesired catalytic quantities of enzyme, the starting material such asglucose, is added to the medium at a level of 1-500 g/L and the culturemaintained at 20° C.° to about 40° C., preferably around 25°-37° C. for1-300 hours until conversion to 2-KLG is effected. The starting materialconcentration may be maintained at a constant level through continuousfeed control, and the 2-KLG produced is recovered from the medium eitherbatchwise or continuously by means known in the art.

C. General Methods Involved in the Invention

In the examples below, the following general procedures were used inconnection with probe construction, screening, hybridization of probe todesired material and in vector construction.

C.1 Isolation of Plasmids, Cleavage with Restriction Enzymes

Plasmids were isolated from the identified cultures using the clearedlysate method of Clewell, D. B. and Helinski, Biochemistry 9: 4428(1970), incorporated herein by reference, and purified by columnchromatography on Biorad A-50 Agarose. Smaller amounts (mini-preps) wereprepared using the procedure of Birnboim, H. C. Nucleic Acids Research7: 1513 (1979).

Fragments of the cloned plasmids were prepared for sequencing bytreating about 20.mu.g of plasmids with 10 to 50 units of theappropriate restriction enzyme or sequence of restriction enzymes inapproximately 600.mu.l solution containing the appropriate buffer forthe restriction enzyme used or sequence of buffers; each enzymeincubation was at 37.degree. C. for one hour. After incubation with eachenzyme, protein was removed and nucleic acids recovered byphenol-chloroform extraction and ethanol precipitation.

As shown in FIG. 1, there are multiple connections the catabolic,pentose and tricarboxylic acids (TCA) pathways. GDH=glucosedehydrogenase; GADH=gluconate dehydrogenase; 2-kDGH=2-keto-d-gluconatedehydrogenase; 2-KDG=2-keto-D-gluconate; IADH=idonate dehydrogenase;2,5-DKG=2,5-diketogluconate; 2KLG=2-keto-L-gulonate;5-KDG=5-keto-D-gluconate; 2KR=2-ketoreductase;2,5DKGR=2,5-diketogluconate reductase; GlkA=glucokinase;GntK=gluconokinase; 5-KR=5-ketoreductase; 2,5-DKGR. As can be seen, theremoval of the glucokinase activity blocks the entry of the glucose intothe catabolic pathway, while the deletion of the gluconokinase activitydoes not allow the entry of gluconate into the pentose assimilatorypathway. From this scheme, it is evident that the absence of theglucokinase and/or gluconokinase activities should impair the metabolismof glucose and its oxidation products.

Production of ASA Intermediates

The present invention provides methods for the production of ascorbicacid intermediates in host cells. The present invention encompassesmethods wherein the levels of an enzymatic activity which phosphorylatesD-glucose at its 6th carbon and/or the levels of an enzymatic activitywhich phosphorylates D-gluconate at its 6th carbon are decreased duringpart or all of the culturing. The present invention encompasses methodswherein the levels of an enzymatic activity which phosphorylatesD-glucose at its 6th carbon and/or the levels of an enzymatic activitywhich phosphorylates D-gluconate at its 6th carbon are increased duringpart or all of the culturing. The present invention also encompasses amethod wherein the levels of an enzymatic activity which phosphorylatesD-glucose as its 6th carbon and/or the levels of an enzymatic activitywhich phosphorylates D-gluconate at its 6th carbon are not modified orare increased at the beginning of the culturing to facilitate growth,that is, to produce cell biomass, and decreased during the later phasesof culturing to facilitate desired product accumulation.

The present invention provides a modified nucleic acid or polynucleotidethat uncouples the oxidative pathway from the catabolic pathway in ahost organism. In one embodiment the modified nucleic acid orpolynucleotide lacks the encoding that expresses the phosphorylation ofthe desired substrate and thus inactivates the phosphorylating enzymaticactivity.

The ASA intermediate may be further converted to a desired end productsuch as ASA or erythorbate. For the production of ASA intermediates, anyhost cell which is capable of converting a carbon source to DKG can beused. Preferred strains of the family Enterobacteriaceae are those thatproduce 2,5-diketo-D-gluconic acid from D-glucose solutions, includingPantoea, are described in Kageyama et al. (1992) International Journalof Systematic Bacteriology vol. 42, p. 203-210. In a preferredembodiment, the host cell is Pantoea citrea having a deletion of part orall of a polynucleotide that encodes an endogenous glucokinase (encodedby nucleic acid as depicted in SEQ ID NO:1) and a deletion of part orall of a polynucleotide that encodes an endogenous gluconokinase(encoded by nucleic acid as depicted in SEQ ID NO:3).

The production of ASA intermediates can proceed in a fermentativeenvironment, that is, in an in vivo environment, or a non-fermentativeenvironment, that is, in an in vitro environment; or combined in vivo/invitro environments. In the methods which are further described infra,the host cell or the in vitro environment further comprise aheterologous genome which has the section encoding the phosphorylationactivity, e.g. the glucokinase sequence and the gluconokinase sequence,inactivated.

A. In Vivo Biocatalytic Environment

The present invention encompasses the use of host cells comprising amodification in a polynucleotide encoding an endogenous enzymaticactivity that phosphorylates D-glucose at its 6th carbon and/or amodification in a polynucleotide encoding an enzymatic activity thatphosphorylates D-gluconate at its 6th carbon in the in vivo productionof ASA intermediates. Biocatalysis begins with culturing the host cellin an environment with a suitable carbon source ordinarily used byEnterobacteriaceae strains, such as a 6 carbon sugar, for example,glucose, or a 6 carbon sugar acid, or combinations of 6 carbon sugarsand/or 6 carbon sugar acids. Other carbon sources include, but are notlimited to galactose, lactose, fructose, or the enzymatic derivatives ofsuch. In addition to an appropriate carbon source, fermentation mediamust contain suitable minerals, salts, cofactors, buffers and othercomponents, known to those of skill in the art for the growth ofcultures and promotion of the enzymatic pathway necessary for productionof desired end-products.

In one illustrative in vivo Pantoea pathway, D-glucose undergoes aseries of membrane oxidative steps through enzymatic conversions, whichmay include the enzymes D-glucose dehydrogenase, D-gluconatedehydrogenase and 2-keto-D-gluconate dehydrogenase to give intermediateswhich may include, but are not limited to GA, KDG, and DKG, see FIG. 1.These intermediates undergo a series of intracellular reducing stepsthrough enzymatic conversions, which may include the enzymes2,5-diketo-D-gluconate reductase (DKGR), 2-keto reductase (2-KR) and5-keto reductase (5-KR) to give end products which include but are notlimited to KLG and IA. In a preferred embodiment of the in vivoenvironment for the production of ASA intermediates, 5-KR activity isdeleted in order to prevent the consumption of IA. Other embodimentshaving other desired inactivated nucleic acid sections depending on thedesired product and desired pathways to be inactivated.

If KLG is a desired intermediate, nucleic acid encoding DKGR isrecombinantly introduced into the Pantoea fermentation strain. Manyspecies have been found to contain DKGR particularly members of theCoryneform group, including the genera Corynebacterium, Brevibacterium,and Arthrobacter.

In some embodiments of the present invention, 2,5-DKGR fromCorynebacterium sp. strain SHS752001 (Grindley et al., 1988, Applied andEnvironmental Microbiology 54: 1770-1775) is recombinantly introducedinto a Pantoea strain. Production of recombinant 2,5 DKG reductase byErwinia herbicola is disclosed in U.S. Pat. No. 5,008,193 to Anderson etal. Other sources of DKG reductase are provided in Table I.

The fermentation may be performed in a batch process or in a continuousprocess. In a batch process, regardless of what is added, all of thebroth is harvested at the same time. In a continuous system, the brothis regularly removed for downstream processing while fresh substrate isadded. The intermediates produced may be recovered from the fermentationbroth by a variety of methods including ion exchange resins, absorptionor ion retardation resins, activated carbon,concentration-crystallization, passage through a membrane, etc.

B. In Vitro Biocatalytic Environment

The invention provides for the biocatalytic production of ASAintermediates, e.g., KDG, DKG and KLG, from a carbon source in an invitro or non-fermentative environment, such as in a bioreactor. Thecells are first cultured for growth and for the non-fermentative processthe carbon source utilized for growth is eliminated, the pH ismaintained at between about pH 4 and about pH 9 and oxygen is present.

Depending upon the desired intermediate being produced, the process mayrequire the presence of enzymatic co-factor. In a preferred embodimentdisclosed herein, the enzymatic co-factor is regenerated. In someembodiments, KDG is the desired ASA intermediate produced, thebioreactor is provided with viable or non-viable Pantoea citrea hostcells comprising a modification in a polynucleotide which normallyencodes an endogenous enzymatic activity that phosphorylates D-glucoseat its 6th carbon and/or a modification in a polynucleotide encoding anenzymatic activity that phosphorylates D-gluconate at its 6th carbon. Inthis embodiment, the host cell also has a mutation in a gene encodingglucokinase (glk) and gluconokinase (gntk) activity. In this embodiment,the carbon source is biocatalytically converted through two oxidativesteps, to KDG, with a reduced loss of substrate, glucose or gluconate,to the catabolic pathway. In this embodiment, there is an increasedyield in the desired intermediate relative the wild-type.

When DKG is the desired ASA intermediate, the bioreactor is providedwith viable or non-viable Pantoea citrea host cells comprising amodification in a polynucleotide encoding an endogenous glucokinase andgluconokinase enzymatic activity that phosphorylates D-glucose at its6th carbon and/or a modification in a polynucleotide encoding anenzymatic activity that phosphorylates D-gluconate at its 6th carbon anda carbon source which is biocatalytically converted through threeoxidative steps, to DKG. In this embodiment, there is no need forco-factor regeneration.

When KLG is the desired ASA intermediate, the bioreactor is providedwith viable or non-viable Pantoea citrea host cells comprising amodification in a polynucleotide which lacks the encoding or is unableto encode an endogenous enzymatic activity that diverts the glucose intothe catabolic pathway, e.g., lacks the ability to phosphorylateD-glucose at its 6th carbon and/or a modification in a polynucleotidewhich lacks the encoding or is unable to encode an enzymatic activitythat phosphorylates D-gluconate at its 6th carbon and a carbon source,such as D-glucose, which is biocatalytically converted through threeoxidative steps, and one reducing step to KLG. In this embodiment, thereductase activity may be encoded by nucleic acid contained within thePantoea citrea host cell or provided exogenously. In this embodiment,the first oxidative enzymatic activity requires an oxidized form of theco-factor and the reducing enzymatic activity requires a reduced form ofco-factor. In a preferred embodiment disclosed herein, the Pantoeacitrea cell is modified to eliminate the naturally occurring GDHactivity and a heterologous GDH activity, such as one obtainable from T.acidophilum, Cryptococcus uniguttalatus or Bacillus species and having aspecificity for NADPH+, is introduced into the Pantoea cell in order toprovide a co-factor recycling system which requires and regenerates oneco-factor. In this embodiment, the host cell further comprises nucleicacid encoding a 2,5-DKG reductase activity or the 2,5-DKG reductase isadded exogenously to the bioreactor.

In another embodiment for making KLG, the bioreactor is charged withPantoea citrea cells comprising a modification in nucleic acid encodingan endogenous enzymatic activity which phosphorylates D-glucose at its6th carbon and/or in nucleic acid encoding an enzymatic activity thatphosphorylates D-gluconate at its 6th carbon and further comprisesnucleic acid encoding membrane-bound GDH, appropriate enzymes andcofactor, and D-gluconic acid is added which is converted to DKG. Thereaction mixture is then made anaerobic and glucose is added. The GDHconverts the glucose to GA, and the reductase converts DKG to KLG, whilecofactor is recycled. When these reactions are completed, oxygen isadded to convert GA to DKG, and the cycles continue.

In the in vitro biocatalytic process, the carbon source and metabolitesthereof proceed through enzymatic oxidation steps or enzymatic oxidationand enzymatic reducing steps which may take place outside of the hostcell intracellular environment and which exploit the enzymatic activityassociated with the host cell and proceed through a pathway to producethe desired ASA intermediate. The enzymatic steps may proceedsequentially or simultaneously within the bioreactor and some have aco-factor requirement in order to produce the desired ASA intermediate.The present invention encompasses an in vitro process wherein the hostcells are treated with an organic substance, such that the cells arenon-viable, yet enzymes remain available for oxidation and reduction ofthe desired carbon source and/or metabolites thereof in the biocatalysisof carbon source to ASA intermediate.

The bioreactor may be performed in a batch process or in a continuousprocess. In a batch system, regardless of what is added, all of thebroth is harvested at the same time. In a continuous system, the brothis regularly removed for downstream processing while fresh substrate isadded. The intermediates produced may be recovered from the fermentationbroth by a variety of methods including ion exchange resins, absorptionor ion retardation resins, activated carbon,concentration-crystallization, passage through a membrane, etc.

In some embodiments, the host cell is permeabilized or lyophilized(Izumi et al., J. Ferment. Technol. 61 (1983) 135-142) as long as thenecessary enzymatic activities remain available to convert the carbonsource or derivatives thereof while reducing the incorporation ofportions of the carbon source or derivatives thereof into the catabolicpathway. The bioreactor may proceed with some enzymatic activities beingprovided exogenously and in an environment wherein solvents or longpolymers are provided which stabilize or increase the enzymaticactivities.

C. Host Cells Producing ASA

Any oxidative or reducing enzymes necessary for directing a host cellcarbohydrate pathway into an ASA intermediate, such as, for example,KDG, DKG or KLG, can be introduced via recombinant DNA techniques knownto those of skill in the art if such enzymes are not naturally occurringin the host cell. Alternatively, enzymes that would hinder an undesiredcatabolic pathway can be inactivated by recombinant DNA methods. Thepresent invention encompasses the recombinant introduction orinactivation of any enzyme or intermediate necessary to achieve adesired pathway.

In some embodiments, Enterobacteriaceae strains that have been cured ofa cryptic plasmid are used in the production of ASA, see PCT WO98/59054.

In some embodiments, the host cell used for the production of an ASAintermediate is Pantoea citrea, for example, ATCC accession number39140. Sources for nucleic acid encoding oxidative or reducing enzymeswhich can be used in the production of ASA intermediates in Pantoeaspecies include the following:

TABLE I ENZYME CITATION glucose dehydrogenase Smith et al. 1989,Biochem. J. 261: 973; Neijssel et al. 1989, Antonie Van Leauvenhoek56(1): 51-61 gluconic acid dehydrogenase Matsushita et al. 1979, J.Biochem. 85: 1173; Kulbe et al. 1987, Ann. N.Y. Acad Sci 6: 5522-keto-D-gluconic acid Stroshane 1977 Biotechnol. BioEng dehydrogenase19(4) 459 2-keto gluconate reductase J. Gen. Microbiol. 1991, 137: 14792,5-diketo-D-gluconic U.S. Pat. Nos: acid reductase 5,795,761;5,376,544; 5,583,025; 4,757,012; 4,758,514; 5,008,193; 5,004,690;5,032,514

D. Recovery of ASA Intermediates

Once produced, the ASA intermediates can be recovered and/or purified byany means known to those of skill in the art, including, lyophilization,crystallization, spray-drying, and electrodialysis, etc. Electrodialysismethods for purifying ASA and ASA intermediates such as KLG aredescribed in for example, U.S. Pat. No. 5,747,306 issued May 5, 1998 andU.S. Pat. No. 4,767,870, issued Aug. 30, 1998. Alternatively, theintermediates can also be formulated directly from the fermentationbroth or bioreactor and granulated or put in a liquid formulation.

KLG produced by a process of the present invention may be furtherconverted to ascorbic acid and the KDG to erythorbate by means known tothose of skill in the art, see for example, Reichstein and Grussner,Helv. Chim. Acta., 17, 311-328 (1934). Four stereoisomers of ascorbicacid are possible: L-ascorbic acid, D-araboascorbic acid (erythorbicacid), which shows vitamin C activity, L-araboascorbic acid, andD-xyloascorbic acid.

E. Assay Conditions

Methods for detection of ASA intermediates, ASA and ASA stereoisomersinclude the use of redox-titration with 2,6 dichloroindophenol (Burtonet al. 1979, J. Assoc. Pub. Analysts 17:105) or other suitable reagents;high-performance liquid chromatography (HPLC) using anion exchange (J.Chrom. 1980, 196:163); and electro-redox procedures (Pachia, 1976, Anal.Chem. 48:364). The skilled artisan will be well aware of controls to beapplied in utilizing these detection methods.

Fermentation Media:

Fermentation media in the present invention must contain suitable carbonsubstrates which will include but are not limited to monosaccharidessuch as glucose, oligosaccharides such as lactose or sucrose,polysaccharides such as starch or cellulose and unpurified mixtures froma renewable feedstocks such as cheese whey permeate, cornsteep liquor,sugar beet molasses, and barley malt. Additionally the carbon substratemay also be one-carbon substrates such as carbon. While it iscontemplated that the source of carbon utilized in the present inventionmay encompass a wide variety of carbon containing substrates and willonly be limited by the choice of organism, the preferred carbonsubstrates include glucose and/or fructose and mixtures thereof. Byusing mixtures of glucose and fructose in combination with the modifiedgenomes described elsewhere in this application, uncoupling of theoxidative pathways from the catabolic pathways allows the use of glucosefor improved yield and conversion to the desired ascorbic acidintermediate while utilizing the fructose to satisfy the metalbolicrequirements of the host cells.

Although it is contemplated that all of the above mentioned carbonsubstrates are suitable in the present invention preferred are thecarbohydrates glucose, fructose or sucrose. The concentration of thecarbon substrate is from about 55% to about 75% on a weight/weightbasis. Preferably, the concentration is from about 60 to about 70% on aweight/weight basis. The inventors most preferably used 60% or 67%glucose.

In addition to an appropriate carbon source, fermentation media mustcontain suitable minerals, salts, vitamins, cofactors and bufferssuitable for the growth or the cultures and promotion of the enzymaticpathway necessary for ascorbic acid intermediate production.

Culture Conditions: Precultures:

Typically cell cultures are grown at 25 to 32 degree. C, and preferablyabout 28 or 29° C. in appropriate media. While the examples describegrowth media used, other exemplary growth media useful in the presentinvention are common commercially prepared media such as Luria Bertani(LB) broth, Sabouraud Dextrose (SD) broth or Yeast medium (YM) broth.Other defined or synthetic growth media may also be used and theappropriate medium for growth of the particular microorganism will beknown by someone skilled in the art of microbiology or fermentationscience.

Suitable pH ranges preferred for the fermentation are between pH 5 to pH8 where pH 7 to pH 7.5 for the seed flasks and between pH 5 to pH 6 forthe reactor vessel.

It will be appreciated by one of skill in the art of fermentationmicrobiology that, now that Applicants have demonstrated the feasibilityof the process of the present invention a number of factors affectingthe fermentation processes may have to be optimized and controlled inorder to maximize the ascorbic acid intermediate production. Many ofthese factors such as pH, carbon source concentration, and dissolvedoxygen levels may affect the enzymatic process depending on the celltypes used for ascorbic acid intermediate production.

Batch and Continuous Fermentations:

The present process employs a fed-batch method of fermentation for itsculture systems. A classical batch fermentation is a closed system wherethe composition of the media is set at the beginning of the fermentationand not subject to artificial alterations during the fermentation. Thus,at the beginning of the fermentation the media is inoculated with thedesired organism or organisms and fermentation is permitted to occuradding nothing to the system. Typically, however, a “batch” fermentationis batch with respect to the addition of carbon source and attempts areoften made at controlling factors such as pH and oxygen concentration.In batch systems the metabolite and biomass compositions of the systemchange constantly up to the time the fermentation is stopped. Withinbatch cultures cells moderate through a static lag phase to a highgrowth log phase and finally to a stationary phase where growth rate isdiminished or halted. If untreated, cells in the stationary phase willeventually die. Cells in log phase generally are responsible for thebulk of production of end product or intermediate.

A variation on the standard batch system is the Fed-Batch system.Fed-Batch fermentation processes are also suitable in the presentinvention and comprise a typical batch system with the exception thatthe substrate is added in increments as the fermentation progresses.Fed-Batch systems are useful when catabolite repression is apt toinhibit the metabolism of the cells and where it is desirable to havelimited amounts of substrate in the media. Measurement of the actualsubstrate concentration in Fed-Batch systems is difficult and istherefore estimated on the basis of the changes of measurable factorssuch as pH, dissolved oxygen and the partial pressure of waste gasessuch as CO.sub.2. Batch and Fed-Batch fermentations are common and wellknown in the art and examples may be found in Brock, supra.

Although the present invention is performed in batch mode it iscontemplated that the method would be adaptable to continuousfermentation methods. Continuous fermentation is an open system where adefined fermentation media is added continuously to a bioreactor and anequal amount of conditioned media is removed simultaneously forprocessing. Continuous fermentation generally maintains the cultures ata constant high density where cells are primarily in log phase growth.

Continuous fermentation allows for the modulation of one factor or anynumber of factors that affect cell growth or end product concentration.For example, one method will maintain a limiting nutrient such as thecarbon source or nitrogen level at a fixed rate and allow all otherparameters to moderate. In other systems a number of factors affectinggrowth can be altered continuously while the cell concentration,measured by media turbidity, is kept constant. Continuous systems striveto maintain steady state growth conditions and thus the cell loss due tomedia being drawn off must be balanced against the cell growth rate inthe fermentation. Methods of modulating nutrients and growth factors forcontinuous fermentation processes as well as techniques for maximizingthe rate of product formation are well known in the art of industrialmicrobiology and a variety of methods are detailed by Brock, supra.

It is contemplated that the present invention may be practiced usingeither batch, fed-batch or continuous processes and that any known modeof fermentation would be suitable. Additionally, it is contemplated thatcells may be immobilized on a substrate as whole cell catalysts andsubjected to fermentation conditions for ascorbic acid intermediateproduction.

Identification and Purification of Ascorbic Acid Intermediates:

Methods for the purification of the desired ascorbic acid intermediatefrom fermentation media are known in the art.

The specific ascorbic acid intermediate may be identified directly bysubmitting the media to high pressure liquid chromatography (HPLC)analysis. Preferred in the present invention is a method wherefermentation media is analyzed on an analytical ion exchange columnusing a mobile phase of 0.01N sulfuric acid in an isocratic fashion.

EXAMPLES General Methods

Materials and Methods suitable for the maintenance and growth ofbacterial cultures were found in Manual of Methods for GeneralBacteriology (Phillipp Gerhardt, R. G. E. Murray, Ralph N. Costilow,Eugene W. Nester, Willis A. Wood, Noel R. Krieg and G. Briggs Phillips,eds), pp. 210-213. American Society for Microbiology, Washington, D.C.or Thomas D. Brock in Biotechnology: A Textbook of IndustrialMicrobiology, Second Edition (1989)

Sinauer Associates, Inc., Sunderland, Mass. All reagents and materialsused for the growth, and of bacterial cells were obtained from DiffcoLaboratories (Detroit, Mich.), Aldrich Chemicals (Milwaukee, Wis.) orSigma Chemical Company (St. Louis, Mo.) unless otherwise specified.

Growth medium for the precultures or inoculuum is commercially availableand preparations such as Luria Bertani (LB) broth, Sabouraud Dextrose(SD) broth or Yeast medium (YM) broth are obtainable from GIBCO/BRL(Gaithersburg, Md.). LB-50 amp is Luria-Bertani broth containing50.mu.g/ml ampicillin.

Fermentation Media:

Two basic fermentation media were prepared for use in the followingexamples, and identified as Seed Flask Media and Fermentation Media.

These basic media were modified by altering the carbon source or by theaddition of other reagents such as sulfite. The reagents useful for therespective media include KH₂PO₄, K₂HPO₄, MgSO₄.7H₂O, Difco Soytone,Sodium citrate, Fructose, (NH₄)₂SO₄, Nicotinic acid, FeCl₃.6H₂O, andtrace salts, including, but not limited to ZnSO₄.7H₂O, MnSO₄.H₂O, andNa₂MoO₄.2H₂O); KH₂PO₄, MgSO₄.7H₂O, (NH₄)₂SO₄, Mono-sodium glutamate,ZnSO₄.7H₂O, MnSO₄.H₂O, Na₂MoO₄.2H₂O, FeCl₃-6H₂O, Choline chloride, MazuDF-204 (an antifoaming agent), Nicotinic acid, Ca-pantothenate and HFCS(42DE). HFCS can also be made according to the desired ratios of glucoseto fructose, e.g., a frucose/glucose solution made of 27.3 g/L powderedfructose, 25.0 g/L powdered glucose.

Cells:

All commercially available cells used in the following examples wereobtained from the ATCC and are identified in the text by their ATCCnumber. Recombinant P. citrea cells (ATCC39140) were used as ascorbicacid intermediate producers and were constructed as described inExamples 4 and 5. Enzymatic assays and genome analysis revealed that thestrains MDP41 and DD6 lacked the genes encoding the glucokinase,gluconokinase and both enzymes whereas the wild-type strains containedgenes encoding the glucokinase and/or gluconokinase enzymes.

Ascorbic Acid Intermediate Analysis:

The presence of ascorbic acid intermediates, e.g., 2-KLG, was verifiedby running a HPLC analysis. Fermentation reactor vessel samples weredrawn off the tank and loaded onto Dionex (Sunnyvale, Calif., ProductNo. 043118) Ion Pac AS10 column (4 mm times 250 mm) connected to aWaters 2690 Separation Module and a Waters 410 DifferentialRefractometer (Milford, Mass.).

Methods of Assaying for Production of Ascorbic Acid Intermediate

Methods for determining the yield, OUR, and CER were described earlierin the definition section.

Recombinant Methods

Vector Sequences

Expression vectors used the methods of the present invention comprise atleast one promoter associated with the enzyme, which promoter isfunctional in the host cell. In one embodiment of the present invention,the promoter is the wild-type promoter for the selected enzyme and inanother embodiment of the present invention, the promoter isheterologous to the enzyme, but still functional in the host cell. Inone embodiment of the present invention, nucleic acid encoding theenzyme is stably integrated into the microorganism genome.

In some embodiments, the expression vector contains a multiple cloningsite cassette which preferably comprises at least one restrictionendonuclease site unique to the vector, to facilitate ease of nucleicacid manipulation. In a preferred embodiment, the vector also comprisesone or more selectable markers. As used herein, the term selectablemarker refers to a gene capable of expression in the host microorganismwhich allows for ease of selection of those hosts containing the vector.Examples of such selectable markers include but are not limited toantibiotics, such as, erythromycin, actinomycin, chloramphenicol andtetracycline.

A preferred plasmid for the recombinant introduction of non-naturallyoccurring enzymes or intermediates into a strain of Enterobacteriaceaeis RSF1010, a mobilizable, but not self transmissible plasmid which hasthe capability to replicate in a broad range of bacterial hosts,including Gram− and Gram+ bacteria. (Frey et al., 1989, The Molecularbiology of IncQ plasmids. In: Thomas (Ed.), Promiscuous Plasmids of GramNegative Bacteria. Academic Press, London, pp. 79-94). Frey et al.(1992, Gene 113:101-106) report on three regions found to affect themobilization properties of RSF1010.

Transformation

General transformation procedures are taught in Current Protocols InMolecular Biology (vol. 1, edited by Ausubel et al., John Wiley & Sons,Inc. 1987, Chapter 9) and include calcium phosphate methods,transformation using DEAE-Dextran and electroporation. A variety oftransformation procedures are known by those of skill in the art forintroducing nucleic acid encoding a desired protein in a given hostcell. A variety of host cells can be used for recombinantly producingthe pathway enzymes to be added exogenously, including bacterial,fungal, mammalian, insect and plant cells.

In some embodiments of the process, the host cell is anEnterobacteriaceae. Included in the group of Enterobacteriaceae areErwinia, Enterobacter, Gluconobacter and Pantoea species. In the presentinvention, a preferred Enterobacteriaceae fermentation strain for theproduction of ASA intermediates is a Pantoea species and in particular,Pantoea citrea. In some embodiments, the host cell is Pantoea citreacomprising pathway enzymes capable of converting a carbon source to KLG.

Identification of Transformants

Whether a host cell has been transformed can be detected by thepresence/absence of marker gene expression which can suggest whether thenucleic acid of interest is present However, its expression should beconfirmed. For example, if the nucleic acid encoding a pathway enzyme isinserted within a marker gene sequence, recombinant cells containing theinsert can be identified by the absence of marker gene function.Alternatively, a marker gene can be placed in tandem with nucleic acidencoding the pathway enzyme under the control of a single promoter.Expression of the marker gene in response to induction or selectionusually indicates expression of the enzyme as well.

Alternatively, host cells which contain the coding sequence for apathway enzyme and express the enzyme may be identified by a variety ofprocedures known to those of skill in the art. These procedures include,but are not limited to, DNA-DNA or DNA-RNA hybridization and proteinbioassay or immunoassay techniques which include membrane-based,solution-based, or chip-based technologies for the detection and/orquantification of the nucleic acid or protein.

Additionally, the presence of the enzyme polynucleotide sequence in ahost microorganism can be detected by DNA-DNA or DNA-RNA hybridizationor amplification using probes, portions or fragments of the enzymepolynucleotide sequences.

The manner and method of carrying out the present invention may be morefully understood by those of skill in the art by reference to thefollowing examples, which examples are not intended in any manner tolimit the scope of the present invention or of the claims directedthereto. All references and patent publications referred to herein arehereby incorporated by reference.

EXAMPLES Example 1 Construction of a Genomic Library from P. citrea139-2a

P. citrea genomic DNA was prepared using the DNA-Pure TM genomic DNAIsolation Kit (CPG, Lincoln Park, N.J.). 50 micrograms of this DNA waspartially digested with the restriction enzyme Sau3A accordingly themanufacturer recommendations (Roche Molecular Biochemicals,Indianapolis, Ind.). The products of the digestion were separated on a1% agarose gel and the DNA fragments of 3-5 kilobases were purified fromthe gel using the Qiaquick Gel extraction kit (Qiagen Inc. Valencia,Calif.). The resulting DNA was ligated with BamH1H-linearized plasmidpBK-CMV (Stratagene, La Jolla, Calif.). A library of around 10xxdifferent plasmids was obtained in this way.

Example 2 β Isolation of the Structural Gene for the Glucokinase Enzyme

To select for a plasmid that carries the glucokinase gene from P.citrea, the genomic library (see above) was transformed into a E. colistrain devoid of the glucokinase gene (glkA) and the PTS transportsystem, strain NF9, glk⁻, (Flores et al., Nat. Biotech. 14, 620-623).After transformation, the cells were selected for growth on M9 mediawith glucose as the only carbon source. With this strategy, plasmidsable to complement the glk⁻ or pts⁻ mutations were selected.

After 48 hrs. of incubation at 37° C., many colonies were visible.Several of these colonies were further purified and their plasmidsisolated and characterized by restriction analysis. It was found thatall the plasmids contained a common DNA fragment.

After re-transforming these plasmids back into NF9, glk⁻, all of themallowed growth on M9-glucose media, corroborating that they were able tocomplement at least one of the mutations present in NF9, glk⁻.

Plasmid pMD4 was isolated in this way and contains an insert of around3.9 kb. The insert in this plasmid was sequenced and it was found thatin a region of around 1010 bp, a gene with a strong similarity to the E.coli glkA gene was present. (SEQ ID 4.)

Example 3 Inactivation of the Glucokinase Gene by HomologousRecombination

The general strategy to inactivate genes by homologous recombinationwith a a suicide vector has been delineated before (Miller andMekalanos., J. Bacteriol. 170 (1988) 2575-2583). To inactivate the glkgene from P. citrea by this approach two plasmids were constructed: pMD5and pMD6.

To construct pMD5, plasmid pMD4 was digested with the NcoI and SnaBlrestriction enzymes accordingly manufacturer specifications. (RocheMolecular Biochemicals, Indianapolis, Ind.). The cohesive ends generatedby these enzymes were blunt-ended with T4 polymerase using standardtechniques. This DNA was ligated with a loxP-Cat-loxP cassette isolatedfrom pLoxCat2 as a SpeI-EcoRV DNA fragment. (Palmeros et al., Gene(2000) 247, 255-264.). This cassette codes for Chloramphenicolresistance. The ligation mixture was transformed into TOP10 competentcell (Invitrogen, Carlsbard Calif.). selecting for growth onChloramphenicol 10 micrograms/ml. Several colonies were obtained after18 hr. incubation at 37° C. The plasmids of some of these colonies werepurified and characterized by restriction analysis. The presence of theloxP-Cat-loxP and the deletion of the DNA region between the NcoI andSnaB1 sites in the glk gene was confirmed. The plasmid with theseproperties was named pMD5.

To construct pMD6, plasmid pMD5 was digested with the BamH1 and Cel11restriction enzymes. The DNA fragment containing the glk geneinterrupted with the loxP-cassette was ligated to a EcoRV-Bsa1 DNAfragment isolated from plasmid pR6Kori1 (unpublished results). Thisfragment contains the R6K origin of replication and the Kanamycinresistance gene. The ligation mixture was transformed into strain SY327(Miller and Mekalanos., ibid.) and transformants were selected on platescontaining kanamycin and chloramphenicol (20 and 10 micrograms/mlrespectively). Several colonies were obtained after 24 hr. incubation at37° C. The plasmids of some of these colonies were purified andcharacterized by restriction analysis. The presence of the loxP-Cat-loxPand the R6K origin was confirmed. The plasmid with these characteristicswas named pMD6.

One characteristic of pMD6 and R6K derivatives in general, is that theycan only replicate in strains that carry the pir gene from plasmid R6K(Miller and Mekalanos., ibid.). P. citrea does not contain the pir geneor sustains replication of pMD6. After transforming pMD6 into P. citrea139-2a and selecting for Cm (R) strains, the proper gene replacement byhomologous recombination was obtained. The inactivation of theglucokinase gene was confirmed by assaying Glucokinase activity usingthe glucokinase-glucose-6-phosphate deydrogenase coupled assay describedby Fukuda et al., (Fukuda Y., Yamaguchi S., Shimosaka M., Murata K. andKimura A. J. Bacteriol. (1983) vol. 156: pp. 922-925). The P. citreastrain where the glucokinase inactivation was confirmed was named MDP4.

Further confirmation of the inactivation of the glucokinase gene wasgenerated by comparing the size PCR products obtained using chromosomalDNA from 139-2a or MDP4 strains and primers that hybridize with theglucokinase structural gene (SEQ. ID. 8, SEQ. ID. 9). With thisapproach, the size of the PCR products should reflect that theloxP-Cat-loxP cassette was cloned in the glucokinase structural gene.

Example 4 Removal of the Chloramphenicol Resistance Marker in MDP4

After overnight growth on YENB medium (0.75% yeast extract, 0.8%nutrient broth) at 30° C., P. citrea MDP40 in a water suspension waselectrotransformed with plasmid pJW168 (. (Palmeros et al., Gene (2000)247, 255-264.). which contained the bacteriophage P1 Cre recombinasegene (IPTG-inducible), a temperature-sensitive pSC101 replicon, and anampicillin resistance gene. Upon outgrowth in SOC medium at 30° C.,transformants were selected at 30° C. (permissive temperature for pJW168replication) on LB agar medium supplemented with carbenicillin (200μg/ml) and IPTG (1 mM). Two serial overnight transfers of pooledcolonies were carried out at 35° C. on fresh LB agar medium supplementedwith carbenicillin and IPTG in order to allow excision of thechromosomal chloramphenicol resistance gene via recombination at theloxP sites mediated by the Cre recombinase (Hoess and Abremski, J. Mol.Biol., 181:351-362). Resultant colonies were replica-plated on to LBagar medium supplemented with carbenicillin and IPTG and LB agarsupplemented with chloramphenicol (12.5 μg/ml) to identify colonies at30° C. that were carbenicillin-resistant and chloramphenicol-sensitiveindicating marker gene removal. An overnight 30° C. culture of one suchcolony was used to inoculate 10 ml of LB medium. Upon growth at 30° C.to OD (600 nm) of 0.6, the culture was incubated at 35° C. overnight.Several dilutions were plated on prewarmed LB agar medium and the platesincubated overnight at 35° C. (the non-permissive temperature for pJW168replication). Resultant colonies were replica-plated on to LB agarmedium and LB agar medium supplemented with carbenicillin (200 μg/ml) toidentify colonies at 30° C. that were carbenicillin-sensitive,indicating loss of plasmid pJW168. One such glK mutant, MDP41, wasfurther analyzed by genomic PCR using primers SEQ ID NO:5 and SEQ IDNO:6 and yielded the expected PCR product (data not shown).

Example 5 Inactivation of the Gluconate Kinase Gene by HomologousRecombination

The general strategy utilized to inactivate the gluconate kinase gene ofP. citrea is presented in FIG. 9, was in essence the same used toinactivate the glucokinase gene as described in example 3. Briefly,after isolating and sequencing a plasmid that allowed a E. coli strain□gntK □idnK, to grow using gluconate as the only carbon source (data notshown); a DNA fragment containing the structural gene for the gluconatekinase gene was generated by PCR using primers SEQ. ID NO: 10 and SEQ.ID NO:11. This approximately 3 kb PCR product was cloned in a multicopyplasmid containing an R6K origin of replication. A unique PstIrestriction site located in the gluconate kinase structural gene asshown in SEQ. ID NO: 2, was utilized to insert a loxP-Cat-loxP cassette.This construction was transferred to the chromosome of the P. citreastrain MDP41 by homologous recombination.

The correct interruption of the gluconate kinase with the loxP-Cat-loxPcassette was confirmed by PCR, using primers SEQ ID NO:11 and SEQ IDNO:12. The new strain, with both glucose and gluconate kinaseinactivated was named MDP5. This strain still contains the Cat markerinserted in the gluconate kinase structural gene. By repeating theprocedure described in example 4, a marker-less strain was obtained andnamed DD6.

Experimental 6

The following illustrates the benefit of a double delete host cell(glucokinase and gluconokinase deleted Pantoea host cells) in terms ofO₂ demand.

Seed Train:

A vial of culture stored in liquid nitrogen is thawed in air and 0.75 mLis added to a sterile 2-L Erlenmeyer flasks containing 500 mL of seedmedium. Flasks are incubated at 29° C. and 250 rpm for 12 hours.Transfer criteria is an OD₅₅₀ greater than 2.5.

Seed Flask Medium

A medium composition was made according to the following:

Component Amount KH₂PO₄ 12.0 g/L K₂HPO₄  4.0 g/L MgSO4•7H₂O  2.0 g/LDifco Soytone  2.0 g/L Sodium citrate  0.1 g/L Fructose  5.0 g/L(NH₄)₂SO₄  1.0 g/L Nicotinic acid 0.02 g/L FeCl₃•6H₂O   5 mL/L (of a 0.4g/L stock solution) Trace salts   5 mL/L (of the following solution:0.58 g/L ZnSO₄•7H₂O, 0.34 g/L MnSO₄•H₂O, 0.48 g/L Na₂MoO₄•2H₂O)

The pH of the medium solution was adjusted to 7.0±0.1 unit with 20%NaOH. Tetracycline HCl was added to a final concentration of 20 mg/L (2mL/L of a 10 g/L stock solution). The resulting medium solution was thenfilter sterilized with a 0.2μ filter unit. The medium was thenautoclaved and 500 mL of the previously autoclaved medium was added to2-L Erlenmeyer flasks.

Production Fermentor

Additions to the reactor vessel prior to sterilization

Component Conc KH₂PO₄ 3.5 g/L MgSO4•7H2O 1.0 g/L (NH₄)₂SO₄ 0.92 g/LMono-sodium glutamate 15.0 g/L ZnSO₄•7H₂O 5.79 mg/L MnSO₄•H₂O 3.44 mg/LNa₂MoO₄•2H₂O 4.70 mg/L FeCl₃•6H₂O 2.20 mg/L Choline chloride 0.112 g/LMazu DF-204 0.167 g/L

The above constituted media was sterilized at 121° C. for 45 minutes.

After tank sterilization, the following additions were made to thefermentation tank:

Component Conc Nicotinic acid 16.8 mg/L Ca-pantothenate 3.36 mg/L HFCS(42DE) 95.5 g/L (gluconate or glucose if desired as the particularstarting substrate)

The final volume after sterilization and addition of post-sterilizationcomponents was 6.0 L. The so prepared tank and medium were inoculatedwith the full entire contents from seed flask prepared as described togive a volume of 6.5 L.

Growth conditions were at 29° C. and pH 6.0. Agitation rate, backpressure, and air flow are adjusted as needed to keep dissolved oxygenabove zero.

Results

The oxidative pathway for ascorbic acid intermediates is depicted inFIG. 10. By determining the amount of carbon dioxide produce (CER), onecan calculate the amount of carbon utilized by the catabolic pathway andthus measure the uncoupling of the catabolic and productive (oxidative)pathways since the sole source of carbon for CO2 is from the carbonsubstrate, no additional CO2 having been supplied into the reactorvessel. When the wild-type organism was utilized in the fermentationprocess, 63% of the glucose was converted to an ascorbic acidintermediate, while 37% was converted, as measured by the CER, tocatabolic products (FIG. 12). In the second phase of the study, thenucleic acid encoding glucokinase expression was run under conditions ofthe wild-type. As shown in FIG. 13A, CO2 evolution decreased to about18%, as measured by CER. Thus glucose catabolism was reduced, but notcompletely uncoupled. In an attempt to ascertain the source, i.e. thepathway wherein the carbon substrate was being diverted to the catabolicpathway, gluconic acid was provided as the sole carbon source. As shownin FIG. 13B in comparison with FIG. 13A, gluconic acid was catabolizedat about the same rate as if glucose had been the carbon substrate. (83%gluconate converted to ascorbic acid intermediate v. 17% of the gluconicacid converted to the catabolic pathway (as measured by CER). See alsoTable 2:

TABLE 2 Fraction of Glucose Fraction of Gluconate converted to convertedto strain Metabolism DKG Metabolism DKG Wild-type 0.37 0.63 — —Glucokinase 0.18 0.82 0.17 0.83 delete (glkA) Gluconokinase 0.24 0.760.02 0.98 delete (gntK)

A last phase of the study was provided by the examination of the OUR andCER of a host cell having the genomic encoding for glucokinase andgluconokinase deleted from the host cell genome. FIG. 14 depicts 3%glucose was converted to CO2, wereas a control (wild-type) exhibited a43% glucose to CO2 yield. As a result, it appears that the wild-typeexhibited a high catabolism of glucose by the catabolic pathway, whichresulted in reduced yield and a high oxygen requirement. However, a dualdeletion of glucokinase and gluconokinase essentially inactivatedcatabolism to less than 10 percent, less than 5 percent and particularly3 or less % of the initial carbon substrate.

Conclusions

The double mutant of glucokinase and gluconokinase appeared to shuntalmost all of the glucose to 2,5-DKG, about 98%.

Example 7 Production of Glycerol from Fructose

To demonstrate that Pantoea citrea can be used to produce chemicalcompounds derived from fructose, glycerol was produced using theapproach described by Empatage et al., [Emptage, M., Haynie, S.,Laffend, L., Pucci, J. and Whited, G. Process for the biologicalproduction of 1,3-propanediol with high titer. Patent: WO 0112833-A 4122-FEB-2001; E.I. DU PONT DE NEMOURS AND COMPANY; GENENCORINTERNATIONAL, INC.]. Briefly, this approach uses two enzymes from yeastto convert dihydroxyacetone phosphate (DHAP) into glycerol as shown inthe reaction depicted in FIG. 25.

The genes for the GPD1 and GPP2 enzymes were cloned in a multicopyplasmid pTrc99 under the control of the Trc promoter (Empatage et al.,2001). This plasmid (pAH48) is able to produce high levels of bothenzymes. The inventors recognized that to produce glycerol in P. citrea,it was desirable to eliminate or reduce the natural ability of thestrain to assimilate glycerol. A common glycerol catabolic pathway inmany bacteria, is through the action of the glycerol kinase [Lin E. C.Ann. Rev. Microbiol. 1976. 30:535-578. Glycerol dissimilation and itsregulation in bacteria]. The inventors found that the P. citrea was ableto grow in media containing glycerols as the only carbon source.Furthermore, inspection of the P. citrea genome sequence, showed that itpossesses a glycerol kinase gene, very similar to the glkA gene from E.coli.

Thus, to eliminate the glycerol kinase activity, the structural gene ofthis enzyme (gene glpK) was inactivated. This was accomplished asdescribed in Examples 3 and 5 (inactivation of glucokinase andgluconokinase genes). Briefly, a 2.9 kb DNA fragment containing theglpKgene and flanking sequences, was obtained by PCR using chromosomalDNA from P. citrea and the primers disclosed in SEQ ID. glpK1 and SEQ ID______. glpK2. This 2.9 kb DNA fragment was cloned in a R6K vector asindicated in Examples 3 and 5. The DNA sequence of the glpKgene is shownin SEQ ID. ______, and the protein sequence of GlpK is shown in SEQ ID.Inspection of the glpK DNA sequence showed the presence of a Hpal site,which was chosen to insert the LoxP-Cat-LoxP cassette. Once the desiredplasmid construction was obtained, the glpK interruption was transferredto the chromosome of P. citrea strain 139-2a ps-, by homologousrecombination as described in example 3 and 5. The resulting P. citreaglpK:: Cm strain was named MDG1.

Once the interruption of the g/pKgene in the P. citrea genome wasconfirmed, the effect of this mutation was evaluated. For such apurpose, strain MDG1 was grown in minimal media M9 containing glycerol0.4% as the only carbon source. After incubating the cells for 48 hoursat 30° C., no growth was observed, indicating that strain MDG1 lost theability to utilize glycerol as a carbon source.

Strain MDG1 was transformed with plasmid pAH48 (Emptage et al., 2001),and the resulting strain MDG2, was tested for its capacity to produceglycerol using fructose as the only carbon source. This was accomplishedby incubating the strain in minimal media containing 2% fructose as theonly carbon source. After incubating the cells for 24 hours at 30° C., asample was collected and analyzed by HPLC as described by Emptage et al.(2001). By doing this, it was found that strain MDG1 did not produce anyglycerol, while strain MDG2 produced 1.36 g/L of glycerol. These resultsdemonstrated that P. citrea was able to divert a substantial part offructose into the formation of glycerol.

Various other examples and modifications of the foregoing descriptionand examples will be apparent to a person skilled in the art afterreading the disclosure without departing from the spirit and scope ofthe invention, and it is intended that all such examples ormodifications be included within the scope of the appended claims. Allpublications and patents referenced herein are hereby incorporated byreference in their entirety.

1. A process for producing an ascorbic acid intermediate in arecombinant host cell comprising, culturing a host cell capable ofproducing said ascorbic acid intermediate in the presence of glucoseunder conditions suitable for the production of said ascorbic acidintermediate wherein said host cell comprises at least one deletionselected from the group of polynucleotides encoding kinases from thegroup consisting of glucokinase and gluconokinase.
 2. The process ofclaim 1 wherein said host cell further comprises both deletions selectedfrom the group of residues encoding kinases selected from the groupconsisting of glucokinase and gluconokinase.
 3. The process of claim 1further comprising the step of recovering said product.
 4. The processof claim 1 further comprising the step of converting ascorbic acidintermediate into a second product.
 5. A method of enhancing theproduction of an ascorbic acid intermediate from a carbon sourcecomprising, d) obtaining an altered Pantoea bacterial strain whichcomprises inactivating a chromosomal gene in a bacterial host strain, e)culturing said altered bacterial host strain under suitable conditions,and f) allowing the production of an ascorbic acid intermediate from thecarbon source, wherein the production of said ascorbic acid intermediateis enhanced compared to the production of the ascorbic acid intermediatein the unaltered bacterial host strain.
 6. The method of claim 5,wherein the ascorbic acid intermediate is selected from the groupconsisting of gluconate, 2-keto-D-gluconate, 2,5-diketo-D-gluconate,2-keto-L-gulonic acid, L-iodonic acid, erythorbic acid, and tartrate. 7.The method of claim 6, wherein the ascorbic acid intermediate is2,5-diketo-D-gluconate.
 8. The method of claim 5, wherein said alteredbacterial strain is obtained by the inactivation of a glk chromosomalgene.
 9. The method of claim 5, wherein said altered bacterial strain isobtained by the inactivation of a gntkchromosomal gene.
 10. The methodof claim 5, wherein said altered bacterial strain is obtained by theinactivation of a glk and a gntk chromosomal gene.
 11. The method ofclaim 5, wherein said bacterial strain is Pantoea.
 12. The method ofclaim 11, wherein said bacterial strain is Pantoea citrea.